We thank the Miami WIHS study participants for their willingness to participate in this work. Data in this manuscript were collected by the Miami Women&#39;s Interagency HIV Study (WIHS). The contents of this publication are solely the responsibility of the authors and do not represent the official views of the National Institutes of Health (NIH). This work was supported by National Institute of Allergy and Infectious Diseases and Women&#39;s Interagency HIV Infection Study (WIHS) [grant number U01 AI103397], National Institute of Allergy and Infectious Diseases, National Institutes of Health [ grant number P30AI073961], National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities, National Institute of Health [grant number UL1TR000460 and 1KL2TR000461], and National Institute of Child Health and Human Development, National Institute of Health [ grant number K23HD074489].

Study activities took place at University of Miami HIV Research Unit in collaboration with the Miami Women&#39;s HIV Interagency Study (WIHS) and the Miami Center for AIDS Research (CFAR).Institutional Review Board (University of Miami Miller School of Medicine) approval was obtained prior to recruitment and any assessment or study related procedures.
1. Endocervical Brush Sample Collection
NOTE: Participants underwent a vaginal examination performed by trained MD or gynecologist and collection of intraepithelial lymphocytes were performed under speculum examination by inserting cytobrush.
<ol>
	<li>Participant characteristics
	<ol>
		<li>Recruit women participants who are aged 18 to 45 years of age, sexually active, not pregnant and not on contraceptive medications or with an intrauterine device.</li>
		<li>Before vaginal examination, have participants undergo a 10 mL blood draw in green top heparin vacutainers for peripheral blood mononuclear cell (PBMC) isolation. to serve as a control for endocervical intraepithelial analysis9.</li>
	</ol>
	</li>
	<li>Collection of the endocervical samples
	<ol>
		<li>Insert a sterile speculum of appropriate size into the vagina without lubrication. Use a warm water to facilitate insertion of the speculum and sterile gauze to clean the mucus. The position of the speculum allows for complete visualization of the os and ectocervix.</li>
		<li>Insert endocervical brush into the endocervical canal until only the bristles closest to the hand are visible. Rotate the brush clockwise for 360&#176;. Transfer the brush into a 15 mL tube containing 5 ml of IMDM medium.</li>
		<li>Immediately after collection, place the tube containing cytobrush on ice. To obtain high viability of collected intraepithelial cells, deliver the cytobrush to the lab and process within 1 h.</li>
	</ol>
	</li>
</ol>
2. Endocervical Cytobrush Sample Processing
<ol>
	<li>Vortex tube containing&#160;endocervical cytobrush applying 4 short (10 sec) strokes.</li>
	<li>Centrifuge the tube, containing cytobrush, for 10 min at 250 x g.</li>
	<li>Carefully remove the cytobrush from the tube (do not disturb the pellet on the bottom of the tube) and add 1 mL of IMDM media and place the tube on ice.</li>
	<li>Place the cytobrush on the 100 mm cell strainer on top of the 50 ml tube.</li>
	<li>Wash the cytobrush with 20 ml IMDM.</li>
	<li>Centrifuge tube for 10 min at 250 x g.</li>
	<li>Decant the supernatant and pelleted cells resuspend in 1 mL IMDM and combine with the already re-suspended pellet described in 2.3.</li>
	<li>Count the cells using an automated cell counter10 or count the cells using hemocytometer by combining 10 &#181;L of the cell suspension with 10 &#181;L of trypan blue dye. Gently pipet up and down ten times to mix the cells and dye. Load 10 &#181;L of the mixture into the opening of either of the two hemocytometer chambers. Count all the cells in the five grid areas. Keep a separate count of the viable and non-viable cells. Determine the cell viability using the following formula: % Viable cells = Non-viable cells x 100/total cell number cells. 
	Determine the subsequent cell concentration per ml (and the total number of cells) using the following calculations. Total Cells per ml = total cells counted x (dilution factor/ number of counted square) x 10,000 cells/ml.</li>
	<li>Freeze isolated endocervical cells using freezing medium containing 10% DMSO and 90% FBS.</li>
</ol>
3. Flow Cytometry
<ol>
	<li>Re-suspend 1-3 x 105 endocervical cells in 100 &#956;L FACS buffer (PBS containing 1% bovine serum albumin</li>
	<li>Add 1 &#956;L/106 LIVE/DEAD Fixable Yellow Dead Cell Stain kit and antibodies for surface markers at the concentrations described in Table 1.</li>
	<li>Incubate the cells for 30 min at +4 &#176;C , protected from light.</li>
	<li>Add 1 mL FACS buffer and the cells and centrifuge tubes for 5 min at 350 x g</li>
	<li>Re-suspend cell pellet in 300 &#956;L 1% paraformaldehyde.</li>
	<li>Prepare compensation controls using beads and antibodies used for staining by adding 100 &#956;L of FACS buffer and one drop of the beads and the same concentration of the antibodies used for sample staining.</li>
	<li>Acquire stained beads and samples on a flow cytometer, equipped with 405 nm, 488 nm, and 635 nm lasers.</li>
</ol>
4. Endocervical Gamma delta T Cell Isolation
<ol>
	<li>Magnetic bead isolation
	<ol>
		<li>Re-suspend endocervical cell pellet (&#8804;107 total cells), described in 2.6, in 40 &#181;L of buffer included in the TCR gamma delta microbead kit referenced in the Table of Materials and Reagents.</li>
		<li>Follow the kit instructions for two-step staining protocol: first, with anti-TCR gamma delta Hapten-Antibody and second, with anti-hapten microbeads-FITC</li>
		<li>After incubation of 15 min at 4-8 &#176;C, wash the cells by adding 1 mL of buffer and centrifuge at 300 x g for 10 min.&#160;Decant the supernatant by inverting the tube, and tap dry with the help of a piece of paper.</li>
		<li>Re-suspend the cell pellet in 500 &#956;L of provided buffer and prepare the magnetic column by placing it in appropriate magnetic separator according to the vendor&#39;s protocol.</li>
		<li>Apply cell suspension onto the column and collect unlabeled cells which pass through and wash column 3x with appropriate amount of buffer (500 &#181;L). Perform washing steps by adding buffer three times, each time once the column reservoir is empty. Collect total effluent. This is the unlabeled, negative cell fraction</li>
		<li>Remove the column form the magnetic separator and pipette 1 ml of buffer onto the column and immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column. 
		NOTE: Isolated gamma delta T cell are already fluorescently stained for flow-cytometric analysis since magnetically labeled Anti-Hapten microbeads are conjugated with fluorescent dye FITC.</li>
	</ol>
	</li>
	<li>Cell sorting
	<ol>
		<li>For cell sorting, label endocervical cells with viability die and antibody panel as described in section 3.</li>
		<li>Set up electronic gates on the live, CD45+ CD3+ and gamma delta positive cells and perform cell sorting.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kaul, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genital+tract+immune+milieu:+an+important+determinant+of+HIV+susceptibility+and+secondary+transmission.">The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission.</a> J Reprod Immunol. 77, 32-40 (2008).</li><li>Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+early+infection+to+prevent+HIV-1+mucosal+transmission.">Targeting early infection to prevent HIV-1 mucosal transmission.</a> Nature. 464, 217-223 (2010).</li><li>Hayday, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma][delta]+cells:+a+right+time+and+a+right+place+for+a+conserved+third+way+of+protection.">gamma][delta] cells: a right time and a right place for a conserved third way of protection.</a> Ann Rev Immunol. 18, 975-1026 (2000).</li><li>Nakasone, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+gamma/delta+T+cells+in+the+lungs+and+their+roles+in+neutrophil-mediated+host+defense+against+pneumococcal+infection.">Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection.</a> Microbes and infection / Institut Pasteur. 9, 251-258 (2007).</li><li>Autran, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+gamma/delta++lymphocyte+subsets+during+HIV+infection.">T cell receptor gamma/delta+ lymphocyte subsets during HIV infection.</a> Clin Exp Immunol. 75, 206-210 (1989).</li><li>Pauza, C. D., Poonia, B., Li, H., Cairo, C., Chaudhry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+Cells+in+HIV+Disease:+Past,+Present,+and+Future.">gammadelta T Cells in HIV Disease: Past, Present, and Future.</a> Front Immunol. 5, 687 (2014).</li><li>Hayday, A. C., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+immunity.">Barrier immunity.</a> Sem Immunol. 21, 99-100 (2009).</li><li>Cheroutre, H., Lambolez, F., Mucida, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+and+dark+sides+of+intestinal+intraepithelial+lymphocytes.">The light and dark sides of intestinal intraepithelial lymphocytes.</a> Nature Rev Immunol. 11, 445-456 (2011).</li><li>Strbo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Intra-Epithelial+Endocervical+Gamma+Delta+(GD)+1+T+Cells+in+HIV-Infected+Women.">Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 T Cells in HIV-Infected Women.</a> AJRI. 75, 134-145 (2016).</li><li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+instrument+for+viability+and+concentration+measurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.</a> Lab Hematol. 10, 109-111 (2004).</li><li>Alcaide, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Vaginosis+Is+Associated+with+Loss+of+Gamma+Delta+T+Cells+in+the+Female+Reproductive+Tract+in+Women+in+the+Miami+Women+Interagency+HIV+Study+(WIHS):+A+Cross+Sectional+Study.">Bacterial Vaginosis Is Associated with Loss of Gamma Delta T Cells in the Female Reproductive Tract in Women in the Miami Women Interagency HIV Study (WIHS): A Cross Sectional Study.</a> PloS one. 11, e0153045 (2016).</li><li>Masson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+genital+tract+cytokine+signatures+of+sexually+transmitted+infections+and+bacterial+vaginosis+in+women+at+high+risk+of+HIV+infection:+a+cross-sectional+study.">Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study.</a> Sex Transm Dis. 90, 580-587 (2014).</li><li>Fichorova, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+(IL)-1,+IL-6,+and+IL-8+predict+mucosal+toxicity+of+vaginal+microbicidal+contraceptives.">Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives.</a> Biol Reprod. 71, 761-769 (2004).</li><li>Juno, J. A., Boily-Larouche, G., Lajoie, J., Fowke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+isolation,+and+flow+cytometric+analysis+of+human+endocervical+samples.">Collection, isolation, and flow cytometric analysis of human endocervical samples.</a> JoVE. , (2014).</li><li>McKinnon, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+viable+leukocyte+sampling+from+the+female+genital+tract+for+clinical+trials:+an+international+multi-site+study.">Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.</a> PloS one. 9, e85675 (2014).</li><li>Dominguez, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiplexed+quantitation+of+gene+expression+on+single+cells.">Highly multiplexed quantitation of gene expression on single cells.</a> J Immunol Methods. 391, 133-145 (2013).</li></ol>

Authors do not have commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding).

Assessment of female lower genital tract mucosal vulnerability is an essential component of clinical studies addressing risk of HIV acquisition and transmission. Typically FGT vulnerability to HIV infection is evaluated by measuring soluble immune modulators in genital secretions (cytokines, chemokines and antimicrobial peptides)12,13. However, these biomarkers are only indicative of vaginal inflammation, are affected by physiological and pathological events, and do not always represent direct mucosal damage. In order to improve our understanding of how mucosal factors increase the risk of acquiring and transmitting HIV, cellular markers of mucosal damage need to be developed. GD intraepithelial T cells are present in the FGT, intercalate between epithelial cells, and are thought to contribute to homeostasis as well as to a first line of defense against environmental challenges. GD T cells have the potential to constitute an additional biomarker to assess mucosal vulnerability in the FGT.
In this protocol, it is shown how to prepare intraepithelial lymphocytes from human endocervical cytobrush sample and is easily mastered. The most critical point of this process is to maintain cell viability by fast processing of the sample as well as by keeping the cells on ice during the entire procedure. The cell yield will depend on cytobrush collection technique, handling skill as well as the infectious status (HIV infection or other viral infections, bacterial or fungal infections). Our group and others have published various studies describing different lymphocyte populations in human genital tract9,14,15. In our hands, one endocervical cytobrush sample can provide 0.5-5 x 106 cells with 80-90% viability. In comparison with the commonly used isolation method14,15, by our method the viability and cell yield is increased.
This method provides a powerful and an efficient tool to study endocervical intraepithelial gamma delta T lymphocytes and could provide insight into other immune cells that migrate to this compartment during infection and/or inflammation. The in vivo cellular composition of mucosal tissues is often difficult to investigate in a comprehensive and quantitative way. Techniques such as immunohistochemistry and flow cytometry are limited by the availability of antigen specific monoclonal antibodies and by the small number of parallel measurements that can be performed on each individual cell. Traditional high-throughput assays, such as gene-expression arrays, when performed on whole tissues, provide information on average gene expression level, and can be only indirectly correlated to quantitative modifications in cellular subpopulations. These limitations become particularly difficult to overcome when studying minority populations, or the cell population that lack exclusive markers. By combining &#34;fluorescence activated cell sorting&#34; (FACS) and &#34;single-cell PCR gene-expression analysis&#34; one can perform a high-throughput transcriptional analysis of the intraepithelial GD T cells in endocervix. This method exploits the capacity of modern flow cytometers to sort individual single cells with accuracy and precision, together with the use of microfluidic technologies to preform high sensitivity multiplexed PCR from minute amounts of mRNA, thereby allowing parallel analysis of the expression of up to 96 genes for each individual cell16.

We developed methodology of using endocervical brush samples to evaluate intraepithelial GD T cells. The endocervical canal is lined by a single layer of columnar epithelium. Intraepithelial lymphocytes represent frontline lymphocytes residing within the epithelial layer that can rapidly initiate immune responses upon encountering pathogenic microbes7,8. Understanding the immunological events of endocervical intraepithelial compartment is important for the design of effective strategies to prevent infections, including HIV.
Our method can be applied for freshly collected human endocervical samples as well as frozen endocervical cells to further explore the role of intraepithelial GD T cells in women with HIV infection or at risk for HIV infection. The main goal of this protocol is to discover novel immunological events in endocervical intraepithelial compartment and to evaluate GD T cell responses as a marker of mucosal vulnerability in women with HIV infection.
The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. Furthermore, the complex heterogeneity of mucosal immune cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the immune system. We developed a method to obtain highly purified intraepithelial GD T cells that can be further analyzed using highly multiplexed, single-cell technologies that may be critical for identifying the underlying mechanisms of FRT immunity.

<table><tbody><tr><td>Cytobrush Plus GT&amp;nbsp;</td><td>(CareFusion, San Diego, CA, USA</td><td></td><td></td></tr><tr><td>IMDM,Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td><td>Beckman Coulter</td><td>496178</td><td></td></tr><tr><td>DMSO, Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>FBS, Fetal Bovine Serum</td><td>Invitrogen, Gibco</td><td>26140-079</td><td></td></tr><tr><td>PBS</td><td>Invitrogen, Gibco</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A-9647</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies,</td><td>L349S9</td><td></td></tr><tr><td>1% paraformaldehyde</td><td>Sigma Aldrich&amp;nbsp;</td><td>D6148</td><td></td></tr><tr><td>Fortessa flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>anti-TCR gamma/delta MicroBead Kit&amp;nbsp;</td><td>Milteny Miltenyi Biotec Inc.</td><td>130-050-701</td><td></td></tr><tr><td>MS column</td><td></td><td>103-042-201</td><td></td></tr><tr><td>MACS separator</td><td></td><td>4124</td><td></td></tr></tbody></table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

Recently, we analyzed endocervical intraepithelial GD T cells and we were the first one to report about the loss of endocervical gamma delta T cells in HIV infected women9,11. Here, we describe the protocol to isolate and analyze the subset of endocervical gamma delta T cells. As shown in Figure 1, GD T cells can be readily detected in the human endocervical cell samples.
A unique population of endocervical gamma delta V1 (GD1) T cells was described using multiparametar flow cytometry-based immunophenotyping (Figure 1). A representative gating strategy for HIV uninfected endocervical sample is shown in Figure 1. Analysis was performed on the total endocervical cells by setting the electronic gate on Forward/Side scatter (Figure 1A) followed by the exclusion of cell doublets (Figure 1B). Viable cells were defined by exclusion of dead cells using LIVE/DEAD Fixable Yellow Amine Dead Cell Stain (Figure 1C). Analysis of CD45 expression was performed on live gated cells (Figure 1D), followed by the analysis of CD3 expression (Figure 1E). Gamma delta 1 (GD1) or gamma delta 2 (GD2) positive cells were defined as a subpopulation of CD3+ cells that express GD TCR (TCR gamma delta V1 or TCR gamma delta V2). Furthermore, it was confirmed that only CD3+ T cells express TCR gamma delta since gated CD3- T cells do not express TCR gamma delta V1 or delta V2. Phenotypic analysis of gated CD3+GD1+ cells revealed (Figure 1G), that majority of GD1 cells (~74%) are CD4-CD8-.
In the present study, feasibility of utilizing endocervical brush samples to purified intraepithelial GD T cells by positive magnetic selection was demonstrated (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/55038/55038fig1.jpg" /> 
Figure 1: Gamma delta (GD) T cell subpopulation in endocervical mucosa analyzed by flow cytometry. Following gating strategy was used to identified endocervical T cell populations: (A) endocervical cytobrush cells were gated according the size and granularity (FSC, forward side scatter vs SSC, side scatter). (B) Cell doublets were excluded by setting the electronic gates in the FSC-A (area) vs SSC-W (width). (C) Live cells are gated on the viability dye-negative population. (D) Electronic gate was set on live, CD45 positive cells. (E) Electronic gate for CD45 positive cells was set on CD3 positive cells (F) TCRVD1 (GD1) and TCRVD2 (GD2) expression was analyzed on CD3+ cells. (G) CD4 and CD8 expression was analyzed on GD1+ cells. (H) TCRVD1 and TCRVD2 expression was analyzed on gated CD3 negative cells. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55038/55038fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55038/55038fig2.jpg" /> 
Figure 2: Isolation of endocervical gamma delta T cells (GD) by positive magnetic separation. Frequency of endocervical GD T cells was confirmed before and after positive magnetic separation. Endocervical cells were first labeled with anti-TCR gamma delta antibody and then with fluorescently stained with Anti-Hapten microbeads-FITC. Percent of TCR GD+ T cells was reported before and after magnetic separation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55038/55038fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Miller School of Medicine, University of Miami. We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Video: Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Medicine

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Cancer Research

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

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